DNA Diagnostic Laboratory - Human and Clinical Genetics (LUMC) - LMDp_Protocols^©

Solutions

(last modified March 31, 2000)

Abbreviations

WFA01: 0.5 M (NH₄)₂SO₄

1. dissolve 6.61 g (NH₄)₂SO₄ in 80 ml milliQ water
2. add milliQ water to 100 ml
3. autovclave
   NOTE: store up to 1 year at RT

WFA09: 10% APS

1. take a vial with 25 g ammonium persulfate (APS) and dissolve it in 200 ml milliQ water
   NOTE: careful, APS is an oxidative substance
2. add milliQ water to 250 ml
3. divide into 10 ml portions in 10 ml blue-cap vials
   NOTE: store maximal 1 year at -20°C or up to 1 month at 4°C

WFA10: ALF loading mix

1. dissolve 0.5 g blue dextran (D-5751, Sigma) in 100 ml de-ionized formamide (WFF01)
   NOTE: formamide is toxic, wear gloves and a lab coat and work in a fume hood
2. warm to dissolve the blue dextran (D-5751, Sigma)
   NOTE: store up to 2 year at 4°C

WFA11: 9% PAA, 80% UF, 0.5 x TAE

1. dissolve 210 g urea in 6.3 ml 50 x TAE-buffer (WFT09), 140 ml 40% acrylamide/bis-acrylamide solution (37.5:1) and 200 ml de-ionized formamide (WFF01)
   NOTE: formamide is toxic, wear gloves and a lab coat and work in a fume hood
   NOTE: use new unopened 100 ml flasks formamide
2. add milliQ water to 625 ml
   NOTE: store up to 3 months at 4°C in a light-tight bottle

WFA12: 9% PAA, 0% UF, 0.5 x TAE

1. add 225 ml 40% acrylamide/bis-acrylamide solution (37.5:1) to 10 ml 50 x TAE-buffer (WFT09)
2. add milliQ water to 1000 ml
   NOTE: store up to 1 year at 4°C in a light-tight bottle

WFB01: 20 x bloodlysis buffer (3.1 M NH₄Cl, 0.2 M KHCO₃, 20 mM EDTA, pH7.4)

1. dissolve 331 g NH₄Cl and 40 g KHCO₃ in 1.6 l milliQ water
2. add 14.8 g EDTA disodium salt and dissolve by heating
3. adjust pH to 7.4 with 5.0 M NaOH (WFN04)
4. add milliQ water to 2 l
5. autoclave
   NOTE: store up to 1 year at RT

NOTE: test the buffer on a spare blood sample (according to ALG001); when a proper leucocyte pellet is obtained the buffer may be used for regular DNA-isolations

WFB04: 10% blocking buffer

1. dissolve 50 g Blocking reagens (#1096176, Boehringer) in 300 ml milliQ water and 50 ml 10 x maleic acid (WFM02)
2. add milliQ water to 500 ml
3. autoclave
4. distribute 8 ml aliquots in 14 ml white-cap tubes.
   NOTE: store max 2 years at -20°C

WFE01: 10 mM EDTA

1. dilute 0.5 M EDTA pH8.0 (15575-012, Life Technologies) 50 times; take 98 ml milliQ water and add 2 ml 0.5 M EDTA pH8.0
2. autoclave
   NOTE: store up to 1 year at RT

WFF01: de-ionized formamide (ABI-310 fragmentanalysis)

1. take a cilinder and add 25 ml (de-ionized) formamide and 1 g mixed bed resin (AG 501X8)
   NOTE: formamide is toxic, wear gloves and a lab coat and work in a fume hood
2. close the cilinder with parafilm and mix 10 min
3. measure the pH using a pH paper
   NOTE: pH must be at least 7.0, but a pH of about 9.0 is optimal. If necessary, repeat the de-ionization step until the desired pH is reached
4. let the resin settle down
5. distribute 500 ul aliquots de-ionized formamide into 1.5 ml Eppendorf tubes and store -20°C
   NOTE: store up to 1 year at -20°C

WFH01: hyb-mix (NaPi/SDS/PEG)

1. freshly prepare 1 l NaPi-buffer;
   1. dissolve 88.9 g Na₂HPO₄.2H₂O in 900 ml milliQ water
   2. adjust the pH to 7.2 using 85% H₃PO₄
   3. add milliQ water to 1 l
      NOTE: prepare fresh, do not store
2. take 1 l NaPi-buffer, add 200 ml 5.0 M NaCl (WFN01) and 8 ml 0.5 M EDTA pH8.0 (15575-012, Life Technologies)
3. add 280 g SDS (Sigma) and dissolve
   NOTE: to weigh SDS, work in a fume hood, wear gloves, a lab coat and a mouth protection
4. add 400 g PEG-6000 and dissolve
   NOTE: if necessary, warm slightly to dissolve
5. add milliQ water to 4 l
6. distribute over 2 flasks of 2 l
   NOTE: store up to 6 months

NOTE: the quality of the hyb-mix should be tested following protocol ALG005 to label a good probe and hybrizing it to an older, working Southern blot. When the signal obtained is OK, the hyb-mix may be used for regular experiments 0.125 M Na₂HPO₄ (pH7.2 with H₃PO₄), 0.25 M NaCl, 1.0 mM Na₂EDTA, 7% SDS (Sigma, 44244), 10% PEG-6,000 (BDH Chemicals Ltd., 44271)

WFK01: nucleus lysis buffer (10 mM M Tris-HCl, 0.4 M NaCl, 2 mM EDTA, pH8.2)

1. dissolve 2.42 g Tris in 1.6 l milliQ water
2. add 8 ml 0.5 M EDTA pH8.0 (15575-012, Life Technologies) and 160 ml 5.0 M NaCl (WFN01)
3. adjust pH to 8.2 with HCl (use glass pipet)
4. add milliQ water to 2 l
   NOTE: store up to 3 months at RT

WFK02: 1.0 M KCl

1. dissolve 7.45 g KCl in 80 ml milliQ water
2. add milliQ water to 100
3. autoclave
   NOTE: store up to 1 year at RT

WFK03: 3.0 M KCl

1. dissolve 18.6 g KCL in 200 ml milliQ water
2. add milliQ water to 250 ml
3. autoclave
   NOTE: store up to 1 year at RT

WFL01: lye-salt solution

1. dissolve 2.4 g NaOH in 150 ml milliQ water
2. add 4 ml 5.0 M NaCl (WFN01)
3. add milliQ water to 200 ml met milliQ water
   NOTE: store up to 1 month at RT

WFL03: 2 x SSCP/STR loading mix

1. take 95 ml (de-ionized) formamide (WFF01)
   NOTE: formamide is toxic, wear gloves and a lab coat and work in a fume hood
2. add 50 mg bromophenolblue (B7021, Sigma) and 50 mg xylene cyanol (XC)
3. add 4 ml 0.50 M EDTA pH8.0 (15575-012, Life Technologies) and mix until both colour stains have dissolved
   NOTE: store up to 2 year at 4°C

WFL04: bacteriophage lambda DNA dilution range

1. prepare a 50 ng/µl bacteriophage lambda DNA dilution by adding 40 µl lambda DNA (stock is 500 ng/µl) to 360 µl autoclaved milliQ water
2. make a dilution range according to the schema below (start with pipetting the SLM-buffer [WFS03] and milliQ water, add lambda DNA last);
   

   Concentration	µl lambda DNA dilution (50 ng/µl)	µl autoclaved milliQ water	µl 2 x SLM-buffer
   1.0 ng/µl	10	240	250
   2.5 ng/µl	25	225	250
   5.0 ng/µl	50	200	250
   10 ng/µl	100	150	250
   20 ng/µl	200	50	250

3. mark the concentration and preparation date on a sticker on the Eppendorf tubes
   NOTE: store up to 1 year at 4°C

WFM01: 1.0 M MgCl₂

1. dissolve 20.33 g MgCl₂.6H₂O in 80 ml milliQ water
2. add milliQ water to 100 ml
3. autoclave
   NOTE: store up to 1 year at RT

WFM02: 10 x maleic acid

1. dissolve 116 g maleic acid (1.0 M) and 87.7 g NaCl (1.5 M) in 900 ml milliQ water
2. adjust the pH to 7.5 with solid NaOH (80-100 g)
   NOTE: the initial white precipitate dissolves at pH7.5
   NOTE: store up to 1 year at 4°C

WFM03: GeneRuler DNA ladder mix (50 ng/µl length marker; 100 bp ladder)

1. add 125 µl DNA ladder mix (0.40 µg/µl) to 775 µl milliQ water
2. take an Eppendorf tube with 10 x SLM-buffer (WFS03), warm until the SDS dissolves and add 100 µl to the diluted DNA ladder mix
3. distribute over two Safe-Lock tubes
   NOTE: store up to 1 year at 4°C

WFM04: bacteriophage lambda HindIII+PstI marker

1. perform two bacteriophage lambda restrition enzyme digestions;
   1. take 500 µg lambda DNA (1.0 ml of a 500 µg/ml stock)
   2. 120 µl 10 x R+ buffer (MBI)
   3. tube 1,  500 U HindIII
      tube 2,  500 U PstI
   4. add milliQ water to 1.2 ml endvolume
2. incubate 4 h at 37°C
3. take 1 µl digested DNA and check on a 0.8 % agarose gel (minimal migration ~10 cm)
   NOTE: if the difestion is incomplete, add 100 U enzym and incubate again 4 h at 37°C
4. if digestion is complete, mix the samples and dilute 10 times in 1 x SLM-buffer, i.e.;
   1. warm a tube with 10 x SLM-buffer (WFS03) to dissolve precipitated SDS
   2. add 1.0 ml 10 x SLM-buffer to the DNA-mixture
   3. add milliQ water to 10.0 ml (i.e. add about 6.6 ml)
5. check the new sample by loading 10 µl on a 0.8 % agarose gel, next to 10 µl of the previously prepared sample
   NOTE: if necessary, modify the dilution
6. distribute the digested samples in 1.0 ml aliquots in marked Safe-Lock tubes
   NOTE: store up to 5 year at -20°C (post-PCR lab)

WFM06: invisible bacteriophage lambda HindIII and PstI marker

1. dilute a bacteriophage lambda DNA HindIII+PstI marker (prepared according to WFM04) to 2 ng/µl;
   1. take 100 µl lambda DNA HindIII+PstI marker
   2. add 0.5 ml 10 x SLM-buffer (WFS03)
   3. add milliQ water to 5.0 ml (i.e. add about 4.4 ml)
2. use 10 µl as marker on a agarose gel for Southern blotting (ALG005)
3. to detect the DNA marker, hybridize with labeled lambda DNA as described (ALG005)
4. distribute the digested samples in 1.0 ml aliquots in marked Safe-Lock tubes
   NOTE: store up to 5 year at -20°C (post-PCR lab)

WFN01: 5.0 M NaCl

1. dissolve 292.2 g NaCl in 800 ml milliQ water
2. add milliQ water to 1 l
3. autoclave
   NOTE: store up to 1 year at RT

WFN02: 6.0 M NaCl

1. dissolve 350.64 g  NaCl in 800 milliQ water (is saturated)
2. add milliQ water to 1 l
3. autoclave
   NOTE: store up to 1 year at RT

WFN03: 3.0 M NaAc (pH 5.6)

1. dissolve 408.1 g NaAc.3H₂O (sodium acetate) in 800 ml milliQ water
2. adjust pH to 5.6 with acetic acid (glacial HAc, about 40 ml, use glass pipet)
3. add milliQ water to 1.0 l
4. re-adjust pH to 5.6 with acetic acid
5. autoclave
   NOTE: store up to 1 year at RT

WFN04: 5 N NaOH

1. dissolve 10 g NaOH in 40 ml milliQ water in a blue-cap tube
2. add milliQ water to 50 ml
   NOTE: store up to 1 month at RT

WFN06: 2 mM dNTP mix (2 mM dATP, dCTP, dGTP and dTTP)

1. pipet 11.5 ml autoclaved milliQ water in a 15 ml tube
   NOTE: use a fresh set of freshly prepared dNTP solutions (100 mM, Life Technologies)
2. add 150 µl of each dNTP
3. mix carefully and distribute 1 ml aliquots to marked 1.5 ml Safe-Lock tubes
   NOTE: store up to 1 year at -20°C

WFN07: 25 mM dNTP mix (25 mM dATP, dCTP, dGTP and dTTP)

1. pipet 250 µl of each dNTP into a 1.5 ml Safe-Lock tube
   NOTE: use a fresh set of freshly prepared dNTP solutions (100 mM, Life Technologies)
2. mix carefully and distribute 100 µl aliquots to marked 1.5 ml Safe-Lock tubes
   NOTE: store up to 1 year at -20°C

WFN08: dNTP mix for radio-active PCR’s (2 mM dATP, dGTP and dTTP, 25 µM dCTP)

1. pipet 11.7 ml autoclaved milliQ water in a 15 ml tube
   NOTE: use a fresh set of freshly prepared dNTP solutions (100 mM, Life Technologies)
2. add 250 µl dATP, dGTP and dTTP solution and 3.1 µl dCTPsolution
3. mix carefully and distribute 1.0 ml aliquots to marked 1.5 ml Safe-Lock tubes
   NOTE: store up to 1 year at -20°C

WFP01: pronase (20 mg/ml)

1. dissolve 1 g pronase (165921, Boehringer Mannheim GmbH) in 50 ml milliQ water (blue-cap Greiner tube)
2. incubate (self digest) 1 h by 37^oC
3. distribute 1.0 ml aliquots to 1.5 ml Safe-Lock tubes
   NOTE: store up to 1 year at -20°C
   NOTE: never refreeze after thawing

WFP02: 5 x multiplex or STR PCR-buffer

1. the 5 x PCR-buffer has final concentrations of 83 mM (NH₄)₂SO₄, 335 mM Tris-HCl pH8.8, 33.5 or 7.5 mM MgCl₂, 34 µM EDTA and 50 mM beta-mercapto ethanol. To prepare the buffers, add to a blue-cap tube;
   

   	multiplex- buffer	STR- buffer
   0.5 M (NH4)2SO4	8.3 ml	8.3 ml
   0.5 M Tris-HCl pH8.8	33.5 ml	33.5 ml
   1.0 M MgCl2	1.675 ml	0.375 ml
   10 mM EDTA	170 µl	170 µl
   14 M beta-mercapto-ethanol	179 µl	179 µl
   autoclaved milliQ water	6.2 ml	7.5 ml

2. distribute 1 ml aliquots to marked 1.5 ml Safe-Lock tubes
   NOTE: store up to 2 year at -20°C

WFP06: 10 x PBS (0.137 mM NaCl / 2.7 mM KCl /8.0 mM Na₂HPO₄ / .. mM KH₂PO₄, non radio-active PTT)

1. dissolve in 1.9 l milliQ water
   • 160 g NaCl
   • 4.0 g KCl
   • 28.8 g Na₂HPO₄.2H₂O
   • 4.8 g KH₂PO₄
2. adjust the pH to 7.5 with 5 N NaOH (i.e. about 10 - 15 ml)
3. add milliQ water to 2 l
4. autoclave
   NOTE: store up to 1 year at RT

WFP07: 6 x PAAGE loading buffer

1. dissolve in 75 ml milliQ water;
   • 0.25 g bromophenolblue (B7021, Sigma)
   • 0.25 g xylene cyanol (XC)
   • 15 g ficoll 400
2. add milliQ water to 100 ml
   NOTE: store up to 2 year at RT

WFS02: 10 x SE-buffer (0.75 M NaCl, 0.24 M EDTA, pH8.0)

1. dissolve 43.8 g NaCl and 89.3 g EDTA disodium salt in 800 ml milliQ water
2. add 11 g NaOH and dissolve
3. adjust pH to 8.0 with 5 N NaOH
4. add milliQ water to 1 l
5. autoclave
   NOTE: store up to 1 year at RT

WFS03: 10 x SLM-buffer

1. dissolve 40 g sucrose (10274 5C, BDH) in 20 ml milliQ water
2. add;
   • 10 ml 10% SDS (15553-019, Life Technologies)
   • 20 ml 0.5 M EDTA (15575-012, Life Technologies)
   • 10 ml 1.0 M Tris-HCl pH7.5 (WFT04)
3. add milliQ water to 100 ml
4. add 250 mg bromophenolblue (B7021, Sigma) and dissolve
   NOTE: store up to 2 year at RT

WFS04: 1.0 M spermidine stock

1. dissolve in a white-cap tube 1 g spermidine (S 0266, Sigma) in 3.0 ml autoclaved milliQ water
2. add milliQ water to 6.9 ml (= 1.0 M stock solution)
3. distribute 1.0 ml aliquots to marked 1.5 ml Safe-Lock tubes
   NOTE: store up to 5 year at -20°C
   NOTE: for 100 mM spermidine take 1 ml 1.0 M spermidine stock, add milliQ water to 10 ml and distribute 200 µl aliquots to marked 1.5 ml Safe-Lock tubes

NOTE: spermidine is agressive and causes burns. Wear protective clothing (lab coat), gloves and protect your eyes. When your eyes come into contact with spermidine, immediately wash with plenty of water and ask for medical advise. Immediately remove any contaminated clothing (see also MSDS)

WFS06: 2 x stopmix

1. weigh 200 mg blue dextran (D-5751, Sigma) and 20 mg phenol red (phenolsulfonphtalein) (P-4758, Sigma)
2. dissolve in 95 ml milliQ water
3. add 5 ml 0.5 M EDTA pH8.0 (15575-012, Life Technologies)
   NOTE: store up to 4 year at RT

WFS08: 10 x Syl PCR-buffer

1. mix in a sterile blue cap tube;
   • 500 µl 0.5 M (NH₄)₂S0₄ (25 mM)
   • 2.0 ml 0.5 M Tris-HCl pH9.6 (100 mM) (WFT04)
   • 1 ml 5.0 M NaCl (500 mM) (WFN01)
   • 1.0 ml 1.0 M MgCl₂ (100 mM) (WFM01)
   • 5.5 ml H₂O
2. distribute 1 ml aliquots in 1.5 ml Safe-Lock tubes
   NOTE: store up to 2 year at -20°C

WFT02: TE-4 (10 mM M Tris-HCl, 0.1 mM EDTA, pH7.5)

1. add 5 ml 1.0 M Tris.HCl pH7.5 (WFT04) and 100 µl 0.5 M EDTA pH 8.0 (15575-012, Life Technologies) to 400 ml milliQ water
2. add milliQ water to 500 ml
3. distribute over 5 bottles with 100 ml each
4. autoclave
   NOTE: store up to 1 year at RT

WFT04: Tris-HCl (0.5 / 1.0 / 1.5 M; pH adjusted)

1. dissolve the required amount of Tris in 80 ml milliQ water;
   • 0.5 M = 6.06 g Tris
   • 1.0 M = 12.12 g Tris
   • 1.5 M = 18.2 g
2. adjust the pH to required figure using HCl (36-38 %), use a glass pipet
3. add milliQ water to 100 ml
4. autoclave
   NOTE: store up to 1 year at 4°C

WFT08: 10 x transferbuffer (non radio-active PTT)

1. dissolve in 1.5 l milliQ water
   • 60.5 g Tris (0.25 M)
   • 288 g glycine (2.0 M)
     NOTE: it might be necessary to warm the solution to dissolve the glycine
2. add milliQ water to 2 l
   NOTE: store up to 1 year at 4°C

WFT09: 50 x TAE-buffer

1. dissolve in 1.0 l milliQ water
   • 484.5 g Tris
   • 272 g NaAc.3H₂O
   • 37.3 g EDTA disodium salt
2. adjust the pH to 8.0 with acetic acid (about 150 ml)
3. add milliQ water to 2 l
4. autoclave
   NOTE: store up to 1 year at RT

WFW01: autoclaved milliQ water

1. fill special 200 ml black capped flasks with milliQ water
2. autoclave
3. mark the flasks with date, content, initials and max. storage date
   NOTE: store up to 1 year at RT
   NOTE: each flask may be opened once during up to 1 year to fill 1.5 ml Safe-Lock tubes

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